The goal of this research is to understand the mechanism of biosynthesis of peptide siderophores in bacteria. Our paradigms are two peptide siderophores produced by the pathogen Vibrio anguillarum. One is anguibactin, an important component of a plasmid-mediated iron uptake system that is essential for virulence of this bacterium. The other is vanchrobactin, a chromosomally-encoded siderophore that in the absence of the pJM1-encoded anguibactin system becomes an important component of the virulence repertoire of V. anguillarum. The specific mechanisms that govern the biosynthesis of these siderophores in this biologically significant pathogen will be further elucidated. In order to dissect these mechanisms we will use a combination of genetic and biochemical approaches. The specific aims are: 1. Dissection of the mechanisms of assembly line enzymology in anguibactin biosynthesis. We plan to ascertain by using in vitro approaches the role of the different domains in each of anguibactin production steps. Specifically, we will assess: the role of the ArCP domain from AngB, the condensation (C) and (PPC) with AngM, the cysteine adenylation domain of AngR in activating cysteine;each Cy domain of AngN in cysteine condensation with 2,3-dihydroxybenzoic acid (DHBA) and cyclization to generate dihydroxyphenylthiazolyn (DHPT);the transfer of DHPT to N-hydroxy- histamine by the C domain of AngM. 2. Do NRPSs communicate with each other through specific domains? Our evidence indicates that several anguibactin biosynthetic genes are encoded redundantly on both the pJM1 plasmid and the chromosome of V. anguillarum and that one, angA is only chromosomally mediated. Our recent work demonstrated that the redundant genes and angA are shared by the biosynthetic apparatus for a chromosomal gene cluster that intervenes in the production of vanchrobactin (N-[N'-(2,3- dihydroxybenzoyl)-arginyl]-serine) and uptake of its ferric complexes and that consists of VabE, VabB, VabD and VabF, as well as other tailoring enzymes. The plasmid-mediated AngB and the chromosomal VabB are NRPSs with two domains each: an isochorysmate lyase and an ArCP that work as the first acceptors of an activated molecule of DHBA in the biosynthesis of anguibactin (AngB) and vanchrobactin (VabB). VabB is slightly larger than AngB due to extensions at the amino and carboxy terminus. Although VabB operates in vanchrobactin biosynthesis it cannot replace AngB in anguibactin biosynthesis synthesis. We believe that these results are due to the presence of specific communication-mediating domains in VabB and AngB that allow them to only interact with their specific NRPSs, VabF for VabB and AngM for AngB. We propose experiments to understand the basis of protein-protein communication between these NRPSs that facilitates the selective interaction in the multienzyme complexes. Our results could lead to the exploration of new avenues in the fascinating field of NRPSs communication during siderophore biosynthesis. These novel approaches will likely lead to a better understanding of siderophore biosynthesis and virulence in bacteria. PUBLIC HEALTH RELEVANCE: We intend to dissect the nonribosomal peptide synthetase systems of pathogenic bacteria into sets of well characterized, interchangeable modular domains that can be used as catalytic reagents for the designed biosynthesis of novel pharmacological and chemotherapeutic products that can be used to combat diseases caused by pathogenic bacteria. These approaches will likely lead to the exploration of new avenues in the process of siderophore biosynthesis and virulence in bacteria.